Storm Eye Institute, Medical University of South Carolina
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Boyer, N. P., Chen, C., Koutalos, Y. Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging. J. Vis. Exp. (52), e2789, doi:10.3791/2789 (2011).
In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca2+ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca2+ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.
Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the photoisomerization of its 11-cis chromophore to all-trans5-7. This regeneration begins with the release of all-trans retinal by the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all- trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).
Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca2+-sensitive fluorescent dyes can be used to examine in detail the interplay between outer segment Ca2+ changes and response to light8-12 as well as the role of inner segment Ca2+ stores in Ca2+ homeostasis13,14. Fluorescent dyes can also be used for measuring Mg2+ concentration15, pH, and as tracers of aqueous and membrane compartments16. Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single photoreceptor cells17-19.
1. Preparation of Sylgard-covered dishes, experimental chambers, and razor blades
2. Preparation of solutions
3. Isolation of retinas
4. Isolation of single photoreceptor cells
5. Fluorescence imaging
6. Representative Results:
Fig. 1 shows the morphology of healthy isolated rod and cone photoreceptors obtained with this protocol from a salamander (Ambystoma tigrinum) retina. Salamander cells have been used extensively for single cell fluorescence imaging studies because of their large size and their ability to survive for several hours after isolation from the retina. In addition, from a salamander retina one can regularly obtain both rod and cone photoreceptors.
One important criterion for the health of the cells is the presence of an intact ellipsoid (Fig. 1), the part of the cell where the mitochondria are concentrated. When the cells are viewed under DAPI optics, this concentration of mitochondria gives a strong fluorescence signal (Fig. 2) due to the presence of NADH. Lack of an intact ellipsoid is a sign of a damaged cell, generally unfit for experiment. Fig. 3 shows a damaged salamander rod photoreceptor, with a swollen cell body and a condensed nucleus. Such cells display much lower fluorescence under DAPI optics, but viewed under FITC optics show a strong FAD signal in the ellipsoid region (originating from oxidized flavin nucleotides and flavoproteins). Another criterion for the health of isolated photoreceptors is their ability to generate all-trans retinol (vitamin A) in their outer segments upon stimulation by light. The generation of vitamin A requires substantial amounts of NADPH, which depends on an intact metabolic machinery. Figures 4 and 5 show the formation of vitamin A in the outer segments of intact frog and mouse rod photoreceptors respectively.
Figure 1. Healthy single rod and cone photoreceptors. The cells were isolated from a tiger salamander retina. Phototransduction takes place in the outer segment and the ellipsoid is densely packed with mitochondria. Rods are responsible for dim light vision, cones for bright light vision.
Figure 2. Fluorescence of living salamander rod and cone. These are dark-adapted cells, showing strong NADH fluorescence in their respective ellipsoids and no significant vitamin A fluorescence in their outer segments. The capture of the fluorescence image represents their first exposure to visible light after the period of dark-adaptation.
Figure 3. Damaged salamander rod photoreceptor. The swollen cell body and the condensed nucleus are indicative of damage. The cell is oxidized and there is minimal NADH signal (DAPI optics), but much stronger FAD signal (FITC optics).
Figure 4. Frog rod with NADH and retinol. This is a healthy frog rod photoreceptor showing strong NADH fluorescence in the ellipsoid region. Before light exposure there is minimal fluorescence in the outer segment. Following light exposure, there is a significant increase in the outer segment fluorescence due to the formation of vitamin A.
Figure 5. Mouse rod with retinol. This is a healthy mouse rod photoreceptor showing significant outer segment fluorescence after light exposure due to the formation of vitamin A. The ellipsoid regions of mouse rod photoreceptors do not show a strong fluorescence signal.
If healthy isolated cells are not obtained, the problem lies either with the isolation or health of the retina or with its chopping. Typically, after removing the front of the eye and the vitreous, the retina readily lifts off the pigment epithelium. If it does not, try to peel it off starting from the periphery of the eyecup. If it is still difficult to separate, a likely possibility is that the animal has not been dark-adapted for an adequate period of time, or the red light is too bright. Ensure proper dark-adaptation for the animal, and dim the red light. The presence of rod photoreceptors in the retina can be easily ascertained by checking the color of the isolated retina: cut a small piece of retina and transfer it to a separate Petri dish, which can then be viewed under room lights. A piece of retina containing rod photoreceptors has a bright red color (due to rhodopsin) that fades rapidly. A colorless piece would indicate the absence of rhodopsin, and hence of rod photoreceptors. This might be due either to improper separation of the retina from the pigment epithelium or to an unhealthy retina. In such case, you should ensure the health of the animals and their proper dark adaptation. If a healthy retina is obtained but not healthy isolated cells, then the problem is most likely with the chopping. A fine chopping is critical: if the chopping is too coarse, or the retina becomes unstuck, it results mostly in pieces of retina instead of isolated cells. Good chopping typically results in a “cloud” of cells appearing in the solution.
Fluorescence imaging of single photoreceptors cells can use endogenous cell fluorophores such as NADH, FAD or vitamin A, as well as fluorescent dyes sensitive to different factors, to probe a wide range of physiological processes in real time. The method can be applied to many different species, including amphibians such as salamander (Ambystoma tigrinum)17,18 and frog (Rana pipiens)20, lizards (Gecko gecko)21, fish (zebrafish, Danio rerio)11, and mouse (Mus musculus)22. The extension of the method to mouse cells allows the study of different types of genetically modified animals.
No conflicts of interest declared.
Supported by NEI grant EY014850.
|Dark room (100-150 ft2)|
|Red lights19||Online stores|
|Infrared light sources andinfrared image viewers||FJW Optical Systems, Inc.|
|Dissecting microscope19||Outfitted with infrared viewers|
|Epifluorescence microscope enclosed in a light-tight cage19|
|Dissecting tools(scissors, forceps, blade holder)||Roboz Surgical Instruments Co.|
|Sylgard elastomer||Essex (Charlotte, NC)||Sylgard 184 elastomer kit|
|Poly-L-lysine (0.1%)||Sigma-Aldrich||P8920||Dilute to 0.01%|
|Experimental chambers||Warner Instruments||D3512P|
|Petri dishes, plastic pipettes||Fisher Scientific|